An RNA virus has RNA (ribonucleic acid) as its genetic material, and infects host cells from bacteria, plants or animals, such as livestock and humans. The major criteria of how RNA viruses are classified are the sense and organization of the viral genome that determines the mode of viral RNA replication, including whether the viral RNA genome has positive (message) or negative sense, whether it is single or double stranded, and whether it is non-segmented or segmented.
Regulatory agencies often require that assays for detection of nucleic acids utilize quality control materials, including standards, calibrators and controls (Molecular Diagnostic Methods for Infectious Diseases; Approved Guideline, 2nd ed., Clinical and Laboratory Standards Institute, vol 26 (8), 2006). Quality control materials insure optimum performance and reliability of test results, including nucleic acid test (NAT) assays. Laboratories are required to demonstrate that assays for detection of viral RNA function properly as intended and are not affected by inhibition or other forms of interference. Controls for qualitative assays provide assurance of true negative and positive results while minimizing the chance for false positive and false negative results. In quantitative assays controls help ensuring accuracy of results. Validation and verification panels are frequently used to check if diagnostic assays or systems (hardware, software and reagents) perform as intended. A panel consists of more than one vial of material useful to improve or check, verify or validate the performance or quality of a diagnostic assay. A panel can, for example, be made from a dilution series that contains different concentrations of an analyte. Calibrators are used to calibrate and recalibrate a diagnostic system, usually after an element of the diagnostic system was changed (e.g. new lot of reagents) or after a predetermined time interval.
Ideally, the quality control material is as similar as possible in structure and morphology to the target analyte so both behave the same when tested. If NAT controls, calibrators or standards behave like a patient sample on different diagnostic systems, they are considered “commutable” amongst these systems. Commutability is a key property of quality control materials that is especially important for calibrators and standards. The quality control material, however, should still be able to generate a signal that is distinguishable from that of the target analyte. Typically, viral RNA assays may have external run controls (positive or negative control) (EC), various types of internal controls (IC), or internal quantification or quantitation standards (QS), as well as calibrators. Internal and external control concepts are further described in CLSI Guideline MM3-A2 and U.S. Pat. Nos. 7,183,084 B2 and 7,192,745.
While PCR and other NAT techniques can test both DNA and RNA, there are technical challenges especially with quality control for RNA assays: 1) RNA is generally more labile than DNA, presenting additional technical difficulties for analytical RNA assays as compared to DNA assays. Naked RNA is sensitive to degradation through RNases, ubiquitously present RNA digesting enzymes. RNases can be found almost everywhere in the environment, however, they are especially prevalent in animal cells and fluids. In order to fully quality control all steps of an RNA assay, it is best to protect the RNA of the quality control material from potential degradation. An intact naturally occurring RNA virus, which does protect its RNA inside the virus, may be used as a calibrator or external positive run control, as long as its RNA sequence contains the primer and probe regions of the target virus. It may not be combined, however, with the test sample to be used as an internal control or quantitative standard, because it would cause a false positive signal. 2) It is desirable that a RNA quality control material should be capable of monitoring the entire diagnostic process and serve as a “full process control”, including nucleic acid isolation, reverse transcription, amplification and detection. 3) The use of materials potentially infectious for humans is not desirable in a diagnostic kit due to safety concerns and shipping regulations. 4) Internal Quality Standards (IQS) like Internal Controls (IC) and internal Quantification or Quantitation standards (QS) materials often can not be obtained from naturally occurring sources. The term “QS” is used in the literature as abbreviation for Quantification Standard, Quantitation Standard, internal Quantification Standard or internal Quantitation Standard, essentially all describing the same type of standard (see Clinical and Laboratory Standards Institute, CLSI Guideline MM3-A2 for details of QS use). This is particularly true for any IQS used as a “competitive” control, which utilizes the same primer sequence as the target RNA, but can be distinguished by a probe sequence different from the target sequence. Such IQS materials usually need to be artificially created. 5) RNA itself is not as amenable to recombinant genetic engineering as DNA and usually requires a DNA intermediate. While it is known to transcribe RNA sequences from recombinant DNA sequences, it is difficult to package and protect these RNA transcript sequences from degradation by RNases.
One approach to solve the issues of RNA instability for quality control materials for RNA viral testing has been the use of “armored RNA,” recombinant RNA packaged in MS2 bacteriophage (e.g., see U.S. Pat. Nos. 5,677,124, and 7,033,749). Armored RNA has a single strand of MS2 RNA containing a recombinant heterologous RNA encapsidated by MS2 bacteriophage proteins to form a pseudo-viral particle.
Armored RNA offers several advantages over naked RNA as a quality control material: 1) It is stable, non-infectious and RNase resistant, and 2) It is useful both as a positive control, an internal control (IC) and quantification standard (QS), and an extraction control. Armored RNA has been useful for the quantification of RNA viruses such as HIV and HCV from human blood. Armored RNA also is utilized as a high titer HCV surrogate material, since it is not possible to grow all different HCV subtypes in culture.
Armored RNA has several disadvantages for use as a quality control material in analytical assays. Ideally, quality control materials should react like the tested analyte in an assay in order to monitor meaningfully all aspects of the procedure. The structure and infected host for armored RNA, however, is very different from that of many animal RNA viruses, e.g. HCV or HIV. A bacteriophage, which infects bacteria, is genetically distant to animal or other eukaryotic viruses. MS2 bacteriophage is not detergent sensitive, because it has a protein coat instead of a lipid bilayer. Many diagnostically relevant enveloped viruses causing harm to humans and livestock (e.g. HIV, Pestiviruses, West Nile Virus (WNV) or HCV) possess detergent sensitive outer envelopes. Because the armored RNA protein coat is very different biochemically from the lipid envelope of these animal viruses, the MS2 bacteriophage particles may behave differently from the targeted animal viral particles in analytical assays. It is conceivable that an Armored RNA QS used to monitor a reaction would fail to indicate that HIV had been destroyed by detergent which contaminated a blood donor test sample. In this case the result could be a false negative blood screening result for HIV resulting in the transfusion of HIV positive CD4 blood cells to several recipients. Armored RNA has never been shown to be commutable, meaning that it behaves like the virus it is supposed to mimic as a quality control. 4) Recovery efficiency of RNA with the most commonly used silica based sample preparation methods is to a certain degree dependent on the length of the RNA. Most human RNA viruses, such as HCV, HIV or WNV, have about three times longer RNA genomes than armored RNA.
Although armored RNA has been useful as a control in analytical assays, it would be preferable to have a quality control material for in vitro diagnostic assays that more closely resembles the targeted animal or human RNA virus than a bacteriophage derived pseudo-viral particle. It would be preferable to have quality control material that is structurally and morphologically similar to the target RNA virus and thus behaves more like e.g. HCV, West Nile Virus (WNV) or HIV viruses. Finally, it would be preferable to have the option of being able to insert heterologous sequences into replication competent viruses.
Chimeric RNA viruses that are similar in structure to the virus being tested would be an ideal quality control material for RNA assays if they were genetically stable and could be grown in culture. In this method, a region of a targeted virus is inserted into the genome of another virus to form a chimeric virus. By testing for the inserted target region, the chimeric virus can function as a quality control material.
In designing a stable viral chimera, it is important to identify a point of insertion in the compact viral genome that does not interfere with the viability of the virus. It is known that the choice of the target region to be inserted, as well as the site for the insertion can dramatically affect chimeric RNA viral functions, especially RNA replication, packaging of the RNA genome, virion stability, and virus infectivity. If the chimeric viral RNA replicates improperly, spontaneous sequence changes, such as deletions or frameshifts, may occur during replication in the RNA sequence of the virus chimera to form useless sequence revertants or pseudo-revertants. The ultimate genomic sequence of the revertant virus is unpredictable and may exclude part or parts of the applied insert. Unstable chimeric RNA virus, therefore, is usually not useful as a quality control material in an analytical RNA assay.
Examples of positive-strand ssRNA chimeras are known that utilize the 5′ nontranslated region (5′NTR) and the open reading frame (ORF). Martin disclosed chimeric GBV-B/HCV (U.S. Pat. No. 7,141,405; US2006/0160067; US2006/0105365). Ilya et al. disclosed chimeric Eastern Equine Encephalitis virus and Sindbis virus (WO 2007/002793). Hong et al. describe HCV/BVDV chimeric constructs where the Npro protease gene is replaced (U.S. Pat. No. 6,326,137). Nam et al. disclosed HCV/BVDV constructs involving exchange of structural genes, especially E1, E2 or C. (U.S. Pat. No. 7,009,044). Rice and Kolykhalov (U.S. Pat. No. 6,127,116) disclosed that functional HCV clones can be used for the assay of HCV by constructing chimeric viruses using components of the IRES, proteases, RNA helicase, polymerase, or 3′NTR to create chimeric derivatives of BVDV whose productive replication is dependent on one or more of these HCV elements. None of these examples, however, disclosed stable Pestivirus RNA chimeras with insertion within the 3′NTR.
While the general concept of chimeric RNA viral constructs is known, no functional examples are provided in the prior art that utilize intentional insertion of heterologous sequences within the 3′NTR of a Pestivirus to obtain a chimeric virus that is replication stable and forms infectious viral particles at titers comparable to that of a wild type virus.
Rice et al. disclosed a concept of constructing BVDV chimeras with inserted sequences from HCV (WO 99/55366; see also Frolov et al., 1998, RNA 4, 1418-1435). No data were given, however, that showed their chimeric constructs involving the 3′NTR were genetically stable. In Example 5 (WO 99/55366), Rice et al described a tandem 3′NTR construct where an HCV 3′NTR insert was placed downstream of the ORF and immediately followed by the intact 3′NTR of BVDV (FIG. 19). Rice et al. reported this 3′NTR HCV-BVDV tandem construct replicated poorly and revertants formed, which showed deletions when sequenced (FIG. 20). Significantly, Rice et al placed the HCV 3′NTR insert precisely downstream of the stop codon of the ORF of BVDV, not within the 3′NTR of BVDV. Rice et al., therefore, did not construct a replication competent BVDV chimera with insertion within the 3′NTR that was genetically stable and could be grown in culture.
Recently, progress has been made in the development of (copy) cDNA clones of full-length BVDV genomes. These so-called “infectious BVDV cDNA clones” allow the in vitro transcription of infectious BVDV RNA genomes (Meyers, et al., J. Virology, 1996, 70: 8606-8613, erratum in J. Virol. 1997, 7 (2): 1735; Vassilev, et al., J. Virology, 1997, 71: 471-478; U.S. Pat. No. 6,001,613).
There is a need in RNA viral testing of Flaviviridae and other RNA viruses to have viral quality control material that is similar in genomic composition and virion composition to the RNA virus being tested.
There is a need in nucleic acid test assays (NAT) for QC materials, standards, calibrators, reference materials, validation and verification material that behave like the target material.
There is a need for such RNA viral quality control materials to be stable in sequence, to be replication stable and to be resistant to degradation by RNases.
It would be useful to have a known region within the 3′NTR of a Flaviviridae/Pestivirus virus defined that allows the stable insertion of heterologous sequences.